ferric-ammonium-citrate and Hemochromatosis

GPR91, a succinate receptor, is expressed in retinal ganglion cells and induces vascular endothelial growth factor (VEGF) expression. RPE also expresses VEGF, but whether this cell expresses GPR91 is not known. Excessive iron is also proangiogenic, and hemochromatosis is associated with iron overload. Therefore, we examined the expression and iron-dependent regulation of GPR91 in the RPE.. GPR91 expression was examined by RT-PCR and immunohistochemistry. Hemochromatosis mice, cytomegalovirus (CMV) infection of retina, expression of CMV-US2 in RPE, and exposure of RPE to ferric ammonium citrate (FAC) were used to examine the iron-dependent regulation of GPR91 expression. VEGF expression was quantified by qPCR. Knockdown of GPR91 in ARPE-19 cells was achieved with shRNA.. GPR91 was expressed in RPE but only in the apical membrane. Retinal expression of GPR91 was higher in hemochromatosis (Hfe(-/-)) mice than in wild-type (WT) mice. Primary RPE cells from Hfe(-/-) mice had increased GPR91 expression compared with WT RPE cells. Iron accumulation in cells induced by CMV infection, expression of CMV-US2, or treatment with FAC increased GPR91 expression. VEGF expression in the Hfe(-/-) mouse retina was increased at ages younger than 18 months, but the expression was downregulated at older ages. The involvement of GPR91 in succinate-induced expression of VEGF in RPE cells was confirmed with GPR91-specific shRNA.. GPR91 is expressed in the RPE with specific localization to the apical membrane, indicating that succinate in the subretinal space serves as the GPR91 agonist. Excessive iron in the retina and RPE enhances GPR91 expression; however, VEGF expression does not always parallel GPR91 expression.

Topics: Animals; Blotting, Western; Cell Line; Female; Ferric Compounds; Gene Expression Regulation; Gene Silencing; Hemochromatosis; Herpesviridae Infections; Humans; Immunohistochemistry; Iron; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Muromegalovirus; Quaternary Ammonium Compounds; Receptors, G-Protein-Coupled; Retinal Pigment Epithelium; Retinitis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Succinic Acid; Transfection; Vascular Endothelial Growth Factor A

Specialized cDNA-based microarrays (IronChips) were developed to investigate complex physiological gene-regulatory patterns in iron metabolism. Approximately 115 human cDNAs were strategically selected to represent genes involved either in iron metabolism or in interlinked pathways (eg, oxidative stress, nitric oxide [NO] metabolism, or copper metabolism), and were immobilized on glass slides. HeLa cells were treated with iron donors or iron chelators, or were subjected to oxidative stress (H(2)O(2)) or NO (sodium nitroprusside). In addition, we generated a stable transgenic HeLa cell line expressing the HFE gene under an inducible promoter. Gene-response patterns were recorded for all of these interrelated experimental stimuli, and analyzed for common and distinct responses that define signal-specific regulatory patterns. The resulting regulatory patterns reveal and define degrees of relationship between distinct signals. Remarkably, the gene responses elicited by the altered expression of the hemochromatosis protein HFE and by pharmacological iron chelation exhibit the highest degree of relatedness, both for iron-regulatory protein (IRP) and non-IRP target genes. This finding suggests that HFE expression directly affects the intracellular chelatable iron pool in the transgenic cell line. Furthermore, cells treated with the iron donors hemin or ferric ammonium citrate display response patterns that permit the identification of the iron-loaded state in both cases, and the discrimination between the sources of iron loading. These findings also demonstrate the broad utility of gene-expression profiling with the IronChip to study iron metabolism and related human diseases.

Topics: Deferoxamine; DNA, Complementary; Ferric Compounds; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HeLa Cells; Hemin; Hemochromatosis; Hemochromatosis Protein; Histocompatibility Antigens Class I; Humans; Hydrogen Peroxide; Iron; Iron Chelating Agents; Membrane Proteins; Nitroprusside; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Promoter Regions, Genetic; Protein Biosynthesis; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; Transfection

The binding of 125I-diferric transferrin to cultured skin fibroblasts and phytohemagglutinin-stimulated lymphocytes was studied in cells derived from individuals homozygous for hereditary hemochromatosis and from normal individuals. Receptors with a high affinity for diferric transferrin were present on all cells. Transferrin receptor number decreased by more than 50% when fibroblasts from both normal and hemochromatotic subjects were maintained in iron-supplemented medium. The number of transferrin receptors expressed by normal and hemochromatotic lymphocytes after mitogen stimulation in iron-supplemented media was less than 50% that of lymphocytes which were mitogen stimulated in standard medium. No change in the affinity of the receptors for diferric transferrin was seen in cells maintained in iron-supplemented medium. Competition experiments in the presence of deferoxamine suggested that the transferrin receptors of fibroblasts and mitogen-stimulated lymphocytes have a 70- to 100-fold higher affinity for diferric transferrin than for apotransferrin. No differences in the properties of transferrin receptors were found between patients with hereditary hemochromatosis and normal individuals. Although transferrin binding decreases when cells are exposed to high levels of iron in the medium, the failure to totally abolish transferrin binding to the receptor suggests that the concentration of diferric transferrin to which cells are exposed may be a major determinant of cellular iron loading in hereditary hemochromatosis.

Topics: Cells, Cultured; Ferric Compounds; Fibroblasts; Hemochromatosis; Humans; Lymphocytes; Quaternary Ammonium Compounds; Receptors, Cell Surface; Receptors, Transferrin; Transferrin